An Evaluation of 20 Years of EU Framework Programme-Funded Immune-Mediated Inflammatory Translational Research in Non-Human Primates

Aging western societies are facing an increasing prevalence of chronic inflammatory and degenerative diseases for which often no effective treatments exist, resulting in increasing health-care expenditure. Despite high investments in drug development, the number of promising new drug candidates decreases. We propose that preclinical research in non-human primates can help to bridge the gap between drug discovery and drug prescription. Translational research covers various stages of drug development of which preclinical efficacy tests in valid animal models is usually the last stage. Preclinical research in non-human primates may be essential in the evaluation of new drugs or therapies when a relevant rodent model is not available. Non-human primate models for life-threatening or severely debilitating diseases in humans are available at the Biomedical Primate Research Centre (BPRC). These have been instrumental in translational research for several decades. In order to stimulate European health research and innovation from bench to bedside, the European Commission has invested heavily in access to non-human primate research for more than 20 years. BPRC has hosted European users in a series of transnational access programs covering a wide range of research areas with the common theme being immune-mediated inflammatory disorders. We present an overview of the results and give an account of the studies performed as part of European Union Framework Programme (EU FP)-funded translational non-human primate research performed at the BPRC. These data illustrate the value of translational non-human primate research for the development of new therapies and emphasize the importance of EU FP funding in drug development

Lymphoid-Like Structures with Distinct B Cell Areas in Kidney Allografts are not Predictive for Graft Rejection. A Non-human Primate Study

Kidney allograft biopsies were analyzed for the presence of B cell clusters/aggregates using CD20 staining. Few B cells were found in the diffuse interstitial infiltrates, but clusters of B cells were found in nodular infiltrates. These nodular infiltrates were smaller shortly after transplantation, and their size increased over time. At the time of clinical rejection, the nodules often presented as tertiary lymphoid structures (TLS) with lymphoid-like follicles. The presence of small B cell clusters during the first 2 months after transplantation was not associated with early rejection. Even in animals that did not reject their allograft, TLS-like structures were present and could disappear over time. Although TLS were more often found in samples with interstitial fibrosis and tubular atrophy (IFTA), TLS were also present in samples without IFTA. The presence and density of clusters resembling tertiary lymphoid structures most likely reflect an ongoing immune response inside the graft and do not necessarily signify a poor graft outcome or IFTA

CNS histopathology.

<p>Shown is the occurrence (number of animals/total number of animals) of perivascular CD3⁺ cell, CD3+ and CD3⁺CD20⁺ (in brackets) clusters in the meninges and perivascular edema with CD68⁺ cells. Positive animals were identified as having several clusters containing >4 positive cells in more than one location.</p

In vivo expansion of T-cells.

<p>Percentages of CD4⁺ T-cells and CD8⁺ T-cells in PBMC, expressed as a percentage of day 0, demonstrate that mainly the CD8⁺ subpopulation is expanded after the infusion of B-LCL, which is consistent with the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071549#pone-0071549-g001" target="_blank">Figure 1</a>. Percentages of CD4⁺ T-cells on day 0 were: 35.7±11.2; 36.8±9.8 and 29.5±19% for groups A, B and C respectively, which is not significantly different from each other (P = 0.4677). Percentages of CD8⁺ T-cells on day 0 were: 18.4±3.5; 21.2±5.2 and 10.2±2.5% for groups A, B and C respectively (P = 0.1451). Consistent with our published data in rhesus monkeys <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071549#pone.0071549-Brok1" target="_blank">[17]</a> an expansion of both CD4⁺ and CD8⁺ T-cells was observed after a booster with MOG_34–56 in IFA.</p

Histological features induced with cMOG_34–56-pulsed B-LCL (group C).

<p>Indicated beneath each panel is the animal and the staining displayed in the respective panel. In all monkeys from group C and in some monkeys from groups A and B perivascular cuffs of infiltrated CD3⁺ T-cells could be found, an example of which is given in panel A. Diffuse infiltrates of CD3⁺ T-cells were more rarely found (B). Only in 3 monkeys lesions of relatively large size were found (C, D and E). (C) Shown is one lesion with PLP staining (C1) with infiltrated CD68⁺ macrophages (C2). The laminin staining in D2 shows that the macrophages remain confined to the Virchow Rubin space and do not pass the glia limitans. The inserts to C2 and D3 show the typical CD68 staining pattern we found. Infiltrates of CD3⁺ and CD20⁺ cells were also found in the meninges, where the two cell types seemed to co-localize (F1, F2). In monkey C4, in which the largest lesions were found, we observed some areas of myelin degeneration (G). It is unclear whether this represents the start of demyelination.</p

Systemic effects of B-LCL infusion.

<p>B-LCL were infused on days 0, 28 and 56. On day 98 animals from groups A (MOG_34–56) and B (CMVmcp_981–1003) were immunized with MOG_34–56 in Incomplete Freund’s adjuvant (IFA). (A) The infusion of autologous B-LCL induces transient increment of circulating lymphocytes. For normalization purposes lymphocyte numbers were expressed relative to day 0 (mean ± SEM 1.8±0.3; 2.4±0.4 and 2.0±0.3×10⁹/l for groups A, B and C respectively, which is not significantly different from each other, P = 0.2184). The increases in numbers of circulating lymphocytes vastly exceeded the number of infused B-LCL. (B) The <i>ex vivo</i> proliferation of unstimulated PBMC was persistently increased after the first B-LCL injection. No significant differences were observed between groups (P = 0.2299). (C) The bodyweights were normalized against the day-0 bodyweights. Bodyweight loss is observed from day 70 onwards, up to 15% in some animals.</p

IgG responses.

*<p>Positive responses developed before the booster immunization on day 98.</p><p>A positive antibody response was defined as the light absorbance at 405 nm being >1.5 times than the absorbance of the day-0 sample on at least two time points.</p

Rhesus monkeys display a naturally occurring cellular immune status against B-LCL.

<p>A) PBMC proliferate <i>ex vivo</i> when cultured with autologous B-LCL (n = 9). The response increased from 835±288 cpm to 16920±3076 cpm (P = 0.0039; Wilcoxon matched pairs test). B) A representative example (animal A4) of the phenotype of proliferating T-cells (percentage cells that have diluted CFSE) present in the natural repertoire proliferating <i>ex vivo</i> against B-LCL. Solid histograms show proliferation in the absence of B-LCL, black lines show proliferation in the presence of B-LCL. C) Mean CFSE dilution in the absence of B-LCL (n = 15; black bars) and in the presence of B-LCL (n = 9). Consistent with the literature on EBV, these are CD3⁺CD8⁺ (regular CTL) and CD3⁻CD56⁺ (NK). Interestingly, also CD3⁺CD8⁺CD56⁺ T-cells proliferate, which are presumably NK-CTL, a subtype that is of interest for the EAE model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071549#pone.0071549-Kap1" target="_blank">[18]</a>.</p

Ex vivo PBMC responses.

<p>(A, B, C) peptide-specific PBMC responses, whereby the background response of PBMC without peptide is subtracted (Δcpm). The background response was 710±177 cpm, but increased to 16000 (1000–39000) cpm (mean (range)) around day 42 to 70, where after it decreased slightly (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071549#pone-0071549-g002" target="_blank">Figure 2B</a>). (D, E, F) PBMC responses in the presence of peptide pulsed autologous B-LCL, whereby the background response of PBMC+non-pulsed B-LCL is subtracted (Δcpm). The background response was between 2700 and 30000 cpm on day zero (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071549#pone-0071549-g001" target="_blank">Figure 1A</a>), but it increased over time. The background response was on average 48000 cpm, and ranged between 10000 and 106000 cpm between days 14 and the end of the experiment. B-LCL were not available in sufficient numbers for all animals for all time points.</p

Improvement of preclinical animal models for autoimmune-mediated disorders via reverse translation of failed therapies

The poor translational validity of autoimmune-mediated inflammatory disease (AIMID) models in inbred and specific pathogen-free (SPF) rodents underlies the high attrition of new treatments for the corresponding human disease. Experimental autoimmune encephalomyelitis (EAE) is a frequently used preclinical AIMID model. We discuss here how crucial information needed for the innovation of current preclinical models can be obtained from postclinical analysis of the nonhuman primate EAE model, highlighting the mechanistic reasons why some therapies fail and others succeed. These new insights can also help identify new targets for treatment